E. coli OMVs induce reactive oxygen species generation and DNA damage. (A) DH5α and LF82 derived OMVs decrease cellular GSH, indicating an increase in ROS. nd denotes not detected. (B) DH5α and LF82 derived OMVs are associated with the formation of mutagenic 8-oxo-dG adducts (green) in Caco-2 nuclei (blue). (C) OMVs induce DNA double strand breaks in Caco-2 cells as measured by Comet assay. (D) OMVs induce the recruitment of γH2Ax histone (green) to sites of damaged nuclear DNA (blue). (E) Flow cytometric analysis of γH2Ax expression during the G1 phase of the cell cycle of Cacco-2 cells. (A), (C) and (E), *, results are significantly different from untreated controls (p ≤ 0.05). ANOVA, followed by two-sided Dunnet’s post hoc test. Results are ± SEM of three independent experiments. (B) and (D), Immunofluorescence microscopy images are from a single representative experiment. Original magnification = 40×, NA1.3 objective used, scale bar indicates 50 μm.